Project Summary/Abstract The present proposal is a revised, competing continuation of AR48120, Post- Translational Modifications in Tolerance and Autoimmunity. Our original proposal detailed a novel property of self proteins that may confer autoimmune responses in murine models of systemic lupus erythematosus (SLE). As introduced in our original proposal, a post-translational protein modification, termed isoaspartyl, can occur spontaneously under physiologic conditions of pH and temperature. While the modification has been known to exist in cells for many years now, the immunity to such modifications within self proteins has only been described by our laboratory. These modifications occur in all cell types and are enhanced in aged and stressed lymphocytes. With relevance to our work, autoantibodies to other protein modifications, notably citrulline proteins, have become diagnostic for autoimmune syndromes such as rheumatoid arthritis. The overall intent of this proposal is to determine how spontanteous biochemical modifications within self proteins can change the immunologic tolerance that is normally established to self proteins. We have identified how two important mechanisms of protein modification can elicit autoimmunity. First, isoaspartyl modified self antigens can undergo altered antigen processing, potentially leading to the expression of novel cryptic self peptides. In particular, histone H2B protein undergoes extensive isoaspartyl modification that may trigger autoimmunity in this manner. Second, an accumulation of intracellular protein modifications is found in the cells of lupus-prone MRL mice causing abnormal T cell hyperproliferation and is coincident with the onset of autoimmune pathology. The present proposal will examine the mechanisms of how isoaspartyl protein modifications alter immunogenicity of proteins and alter lymphocyte functions. In particular, we will develop novel mouse strains with conditional protein repair systems that alter protein modification in CD4 T cells. We will determine how autoantigen processing is altered in the presence of protein modification and how modified histone protein triggers autoantibody responses. The overall goal of these studies are to identify the roles of posttranslational protein modifications in the genesis of lupus-like autoimmunity. Project Narrative Systemic autoimmune diseases are characterized by aberrant immune responses directed at a select group in intracellular proteins. The focus of our work is to identify novel posttranslational protein modifications that may be critical in the induction of autoimmune disease. Precedence for these studies is apparent by the diagnostic and clinical relevance of several protein modifications, notably citrulline, in rheumatic disease. The present studies focus on a protein modification termed `isoaspartyl' that occurs spontaneously in eukaryotic cells and is enhanced in conditions of cellular stress and inflammation. We have previously identified the presence of isoaspartyl modifications in two lupus autoantigens, the snRNP ribonucleoprotein and histone H2B. We will examine the immunogenicity that arises in the context of this modification and determine how the course of autoimmunity is altered when isoaspartyl protein modifications are repaired in vivo. The goal of this work is to determine the importance of spontaneous protein modification in B and T cell immune tolerance and autoimmune pathology.